ABSTRACT Primary immune deficiencies with impaired cell-mediated cytotoxicity commonly manifest as Familial Hemophagocytic Lymphohysticytosis (FHL), a life threatening disorder. FHL is characterized by disproportioned immune response mediated by excessively activated Cytotoxic T-lymphocyte (CTLs), Natural Killer (NK) and macrophages. FHL subjects usually harbor biallelic germline-mutations in genes involved in intracellular membrane trafficking including Syntaxin 11 (STX11, FHL-4) and Syntaxin-Binding Protein 2 (STXBP2, FHL-5). Nonetheless, monoallelic mutations have also been associated in FHL, but their clinical and biological significance remain unclear. Despite evidences linking STX11 and STXBP2 mutations to abnormal CTL and NK cell activity in FHL patients, there is a significant gap in knowledge about the normal function of these proteins and how these mutations impact on cytotoxic activity and ultimately lead to disease. This proposal aims to bridge this gap in knowledge between the genetic findings and the clinical manifestations in FHL-patients by establishing the basic mechanisms through which STX11/STXBP2 cooperate to effect LG exocytosis and by dissecting how they are affected in patient cells. STX11 and STXBP2 physically interact in as yet poorly characterized ways to control the release of lytic granule (LG) content at the immunological synapse. Data from our lab provide evidences that STX11 also interact with different sets of membrane fusion proteins that could mediate distinct trafficking steps. We hypothesize that STX11/STXBP2 control endosome- lysosome trafficking and LG fusion at the plasma membrane through these interactions. We further hypothesize that FHL mutations interfere with these interactions and thus impair cell-mediated cytotoxicity and/or survival signaling, a process that ultimately results in an array of immunological disorders with HLH symptoms. First, we will evaluate how FHL-mutations in STX11 and STXBP2 affect the physical relationship with one another and with other interacting proteins involved in exocytic and endo-lysosomal pathways by using biochemical and Mass Spec approaches in patient cells. We will establish how mutations affect the structural requirements for protein-protein interactions by performing pull-down, liposome co-flotation assays and surface plasmon resonance experiments. Second, we will assess the biological significance of both monollelic and biallelic FHL-mutations by investigating the intracellular trafficking defects in FHL-patient cells using super resolution STED and TIRF microscopy. Moreover, we will establish the molecular mechanisms of action of these mutations by using a unique in-vitro fusion assay develop in our lab. In summary, our studies will contribute more broadly to our understanding of the membrane trafficking steps that control cell killing pathways in CTL and NK cells, help in therapeutic decisions for patients carrying monoallelic mutations, establish common and gene-specific pathways affected in wide range of genetic disorders that share some manifestations of HLH symptoms and aid in the development of new tools for rapid diagnostic.